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Contents
Problem
Possible Cause
Suggestions
L ow A 2 6 0 /A 2 8 0
Extended
Resin from the column may be
ratio
centrifugation during
pre sen t in e lua te. A void
elution step.
centrifugation at speeds higher than
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Storage and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
specified. The material can be
removed from the eluate by
centrifuga tion — it w ill not interfere
with PCR or restriction digests.
Hemoglobin remains
After app lication of sam ple to
o n c olu m n
column, wash once with 300 : l Buffer
Binding Capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Before Starting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
DCL.
No DNA eluted
Poor ce ll lysis due to
Mix thoroughly with Buffer DCL prior
impro per m ixing with
to loading H iBind™ co lumn .
Buffer DCL
B. Ciuculating DNA Vacuum Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
No eth anol add ed to
Dilute DNA Wash Buffer with the
Wash Buffer
indicated volume of absolute ethanol
Concentrate.
before use.
Incomplete lysis due
Bu ffer DC L is vis cou s an d th e sa m ple
colored residue
to improper mixing
mu st be m ixed tho rough ly.
in co lu m n
with Buffer DCL
Washing leaves
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A. Circulating DNA Spin Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
No eth anol add ed to
Dilute DNA Wash Buffer with the
DNA Buffer
indicated volume of absolute ethanol
Concentrate.
before use.
Determination of Yield and Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
B. Vacuum Protocol: Purification of Circulating DNA from Plasma
or Serum
Kit Contents
Product No.
Purification times
D3091-00
D3091-01
D3091-02
5 Preps
50 Preps
200 Preps
5
50
200
15
150
600
Material and equipments supplied by user
#
Tabletop microcentrifuge and sterile 1.5 ml tubes
#
Vacuum Manifold
2 ml Collection Tubes
1.
Buffer DCL
10 ml
50 ml
200 ml
Buffer ACB
20 ml
100 ml
2 x 200 ml
5 ml
50 ml
150 ml
310 :g
310 :g
2 x 1 mg
DNA Wash Buffer
5 ml
20 ml
3 x 20 ml
Nuclease-Free Water
5 ml
40 ml
160 ml
OB Protease
600 :l
6 ml
24 ml
User Manual
1
1
1
2.
Prepare the lysate by following step 1-6 of Protocol A, Spin protocol on page
4.
Insert the HiBind® DNA Micro column into the vacuum manifold. Carefully apply
the lysate to an HiBind® DNA column. Turn on the vacuum source to draw all
liquid through the column. When all lysates have been drawn through the
column completely, switch off the vacuum pump.
Note: If the lysate has difficulty to pass through the column at this stage. Place
the column into a collection tube (supplied). Close the lid and centrifuge at
8000 x g for 5 minutes or until all liquid pass through the column. Place the
column into another collection tube (supplied) and continue step 10 of the spin
protocol.
3.
4.
5.
6.
7.
Pipet 700 :l of Buffer ACW1 into the column. Turn on the vacuum source to
draw all liquid through the column. Turn off the vacuum.
HiBind™ DNA Micro columns
Buffer ACW1
Carrier RNA
Before Starting
Reconstitute Carrier RNA in Nuclease-Free Water at
final concentration of 1mg/ml. Vortex vial briefly prior
1
to use. We recommend that you aliquot and store vials
of reconstituted protease at -80oC.
Wash the column by pipetting 700 :l of DNA Wash Buffer diluted with
ethanol into the column. Turn on the vacuum source to draw all liquid through
the column. Turn off the vacuum.
Wash the column by pipetting another 700 :l of DNA Wash Buffer diluted
with ethanol into the column. Turn on the vacuum source to draw all liquid
through the column. Turn off the vacuum.
IMPORTANT
DNA Wash Buffer Concentrate must be diluted with
absolute ethanol (96-100%) as follows:
2 D3091-00
Add 20 ml absolute ethanol
D3091-01
Add 80 ml absolute ethanol
D3091-02
Add 80 ml absolute ethanol per bottle
®
Close the lid of HiBind DNA column, remove it from the vacuum manifold.
Insert the column into a collection tube (supplied) and centrifuge at 13,000 x
g for 5 minute to completely dry the column.
Store diluted DNA Wash Buffer at room temperature
Elute DNA as Step 14-15 on page 5.
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Introduction
Determ ination of Yield and Quality
E.Z.N.A.® Circulating DNA Kit provides a rapid and easy method for the isolation of
Circulating DNA from plasma, serum, and other cell-free body fluids. Samples can be
either fresh or frozen, provided that they have not been frozen and thawed more
than once. The kit allows single or multiple, simultaneous processing of samples in
under 120 minutes. There is no need for phenol/chloroform extractions, and timeconsuming steps such as CsCl gradient ultracentrifugation, and precipitation with
isopropanol or ethanol, are eliminated. DNA purified using the E.Z.N.A® Circulating
DNA method is ready for applications such as PCR, Circulating detection, and
genotyping.
E.Z.N.A.® Circulating DNA Kit uses the reversible nucleic acid-binding properties of
HiBind® matrix, combined with the speed of mini-column spin technology. A
specifically formulated buffer system allows Circulating DNA bind to the matrix.
Samples are first lysed under denaturing conditions and then applied to the HiBind®
DNA spin columns to which DNA binds, while cellular debris, hemoglobin, and other
proteins are effectively washed away. High quality DNA is finally eluted in sterile
deionized water or low salt buffer.
The total DNA yield can be determined by a spectrophotometer using deionized
water, Tris-HCl buffer, or Elution Buffer as blank. Dilute the DNA in TE buffer and
calculate concentration as:
[DNA] = (Absorbance260) x (0.05 :g/:l) x (Dilution factor)
The quality of DNA can be assessed by measuring absorbance at both 260 nm and at
280 nm. A ratio of (A260/A280) of 1.7-1.9 corresponds to 85%-95% purity.
Troubleshooting Guide
Problem
Possible Cause
Suggestions
C lo gg ed Co lu m n
Inco m ple te ly sis
Add the correct volume of Buffer DCL
and incubate for specified time at
70 o C. It may be necessary to extend
incubation time by 10 min.
Storage and Stability
Sample too viscous
Divide sample into mu ltiple tubes,
adjust volume to 250 : l w it h 1 0 m M
®
All components of the E.Z.N.A. Circulating DNA Kit, except the Carrier RNA should
be stored at 22oC-25oC. Once reconstituted in water, Carrier RNA should be stored at
-20oC. For long-term storage, OB Protease can be stored at 4oC. Under these
conditions, DNA has successfully been purified and used for PCR after 24 months of
storage. Under cool ambient conditions, a precipitate may form in the Buffer DCL
and ACB. In case of such an event, heat the bottle at 37oC to dissolve. Store Buffer
DCL and ACB at room temperature.
Expiration Date: All E.Z.N.A.® Circulating DNA Kit components are guaranteed for at
least 24 months from the date of purchase when stored at 22-25oC.
Tris -HC l.
Low DN A yie ld
C lo gg ed co lu m n
See above
Poor elution
Repeat elution or increase elution
volum e (see n ote on p age 4).
Incubation of column at 70 o C for 5
min with Nucleic-free water may
increase yields.
Improper washing
DNA W ash Buffer Concen trate must
be diluted with absolute (100%)
ethanol as specified on page 5
before use.
Binding Capacity
Each HiBind
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®
column can bind approximately 20 :g DNA.
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A. Spin Protocol: Purification of Circulating DNA from 0.1-1ml Plasma or
Serum
Materials and equipments Supplied by User
#
Tabletop microcentrifuge and sterile 1.5 ml tubes.
#
Water bath - set to 60oC.
11. Place the column into a same 2 ml tube from step 10 and wash by pipetting
700 :l of DNA Wash Buffer diluted with ethanol. Centrifuge at 8,000 x g for 1
min. Again, dispose of collection tube and flow-through liquid.
NOTE: The procedure below has been optimized for use with FRESH or FROZEN
Plasma or Serum samples from 0.1 to 1 ml in volume. Other Cell-free samples can
also be used. For DNA extraction from Blood, we suggest using the E.Z.N.A.® Blood
DNA Kit (product number D3392). To isolate Circulating RNA from serum or other
non-cellular body fluids use E.Z.N.A.® Circulating RNA Kit.
Preheat an aliquot of Nuclease-Free Water (approximately 0.1 ml per sample) at
60oC. Carry out all centrifugation steps at room temperature.
1.
Add # 25 :l OB Protease, ! 50 :l OB Protease, or
a sterile microcentrifuge tube.
2.
Add # 250:l, ! 500:l, or
Protease.
3.
a 100 :l OB Protease to
Note that DNA Wash Buffer is provided as a concentrate and must be diluted
with absolute ethanol as indicated on the bottle or page 3. If refrigerated,
the diluted wash buffer must be brought to room temperature before use.
12. Using a new collection tube, wash the column with a second 700 :l of DNA
Wash Buffer and centrifuge as above. Discard flow-through and re-use the
collection tube for next step.
13. Place the empty column into the same 2 ml collection tube form step 12,
centrifuge at maximum speed (13,000 x g) for 5 min to dry the column. This
step is crucial for ensuring optimal elution in the following step.
a 1ml plasma or Serum to the tube containing OB
Add # 200:l DCL Buffer and 5.6 :l Carrier RNA, ! 400 :l DCL Buffer and 5.6
:l Carrier RNA, or a 0.8ml Buffer DCL and 5.6 :l Carrier RNA to the sample.
Vortex at maxi speed for 30s to mix thoroughly.
4.
Incubate at 60oC for 30 min.
5.
Add # 450 :l ACB Buffer, ! 900 :l ACB Buffer, or
Vortex at maxi speed for 30s to mix thoroughly.
6.
Incubate the mixture on ice for 5 min.
7.
Assemble an HiBind® DNA Micro column in a 2 ml collection tube (provided).
8.
Transfer 750 :l of the lysate from step 6 into the column. Centrifuge at
8,000 x g for 1 min to bind DNA. Discard flow-through liquid and assemble the
column into the same collection tube.
9.
Repeat step 8 until all of the lysate pass through the Micro column.
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10. Place the column into a second 2 ml tube (provided) and wash by pipetting
700:l of Buffer ACW1. Centrifuge at 8,000 x g for 1 min. Again, Discard flowthrough liquid and reuse the collection tube for next step.
14. Place the column into a sterile 1.5 ml microfuge tube and add 20-150 :l of
preheated (60oC) Nuclease-Free Water. Allow the columns to sit for 5 min at
room temperature.
15. To elute DNA from the column, centrifuge at 8,000 x g for 1 min. Store the
eluted DNA at -20°C.
a1.8 ml Buffer ACB.
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